Synthetic genetic feedback controller circuits to reprogram cell fate PI: Domitilla Del Vecchio1;4 co-PIs: James J. Collins2;4;5;6, Thorsten Schlaeger7, and Ron Weiss2;3;4 1Department of Mechanical Engineering, MIT; 2Department of Biological Engineering, MIT 3Department of Electrical Engineering and Computer Science, MIT 4Synthetic Biology Center, MIT; 5Broad Institute of MIT & Harvard; 6The Wyss Institute 7 Stem Cell Transplantation Program, Boston Children's Hospital PROJECT SUMMARY The past decade has seen monumental discoveries in the stem cell ?eld, with demonstrations that the fate of a terminally differentiated cell, contrary to what was traditionally believed, could be reverted back to pluripotency or directly converted to other differentiated cell types. All of a sudden, new approaches to regenerative medicine seem within reach: lost or damaged cells could be replaced by patient-speci?c reprogrammed cells, thus providing on- demand, compatible, high-quality cells of any required type. To meet this vision, the scienti?c community has made tremendous efforts toward establishing robust and ef?cient protocols for cell fate reprogramming. These protocols are largely based on a priori ?xed (pre?xed) ectopic overexpression of suitable transcription factors (TFs), with the rationale that this overexpression could trigger transitions among the states of the gene regulatory networks (GRNs) that take part in cell fate determination. Yet, despite a decade of remarkable progress, the ef?ciency of these protocols remains low, the quality of produced cells is often unsatisfactory, and many potentially useful direct cell fate conversions still seem impossible. These issues pose a formidable obstacle to the practical use of both human induced pluripotent stem cells (hiPSCs) and transdifferentiated cells in regenerative medicine. Arguably, our ability to accurately and precisely steer the concentrations of GRNs' TFs within desired ranges is critical to the success of cell fate reprogramming. Unfortunately, current protocols based on pre?xed TFs' overexpression have not demonstrated this critical ability. To address this problem, we propose a completely new approach to cell fate reprogramming in this project: we replace pre?xed overexpression with feedback overexpression of TFs, which we realize with an in vivo synthetic genetic feedback controller circuit. Within this circuit, the overexpression level is not a priori ?xed and is adjusted based on the discrepancy between desired and actual TF's concentrations. It therefore can accurately and precisely control TFs' concentrations to desired values, independent of the endogenous GRN that also regulates these TFs. Our research plan focuses ?rst on hiPSC reprogramming as a test-bed for evaluating the bene?t of our approach and second on directed differentiation of hiPSCs into platelets as a directly clinically relevant application. Speci?cally, in AIM 1, we propose to systematically investigate the ef?cacy of pre?xed overexpression of pluripotency TFs for hiPSC reprogramming. In AIM 2, we propose to construct and test the synthetic genetic feedback controller circuits that implement feedback overexpression of a number of TFs concurrently. In AIM 3, we will leverage the synthetic genetic feedback controller circuits for human hiPSC reprogramming and for directed differentiation of hiPSCs into platelets. This project will result in substantially higher reprogramming ef?ciencies, in cell products that more closely resemble the target cell type, and in the future, in cell conversions that today seem not possible. More broadly, our synthetic genetic feedback controllers will empower scientists and practitioners with a new tool to accurately control the TFs' concentrations of any endogenous GRNs and, in particular, of those GRNs involved in cell fate determination. 1